Abstract: Lu’an Guapian is one of the varieties of Chinese green tea. Conventional processing can often require only single fresh leaves while excluding buds and stems. However, mechanical harvesting can inevitably mix the leaves at varying maturity stages within the raw material, leading to the low consistency and stability of the final product. Therefore, the optimal grading and processing techniques are essential to clarify the biochemical and sensory alterations induced by leaf maturity. This study aims to explore in vitro bioactivities and taste characteristics of Lu’an Guapian tea using leaf grade differences. The tea samples were prepared from fresh leaves harvested at three positions relative to the bud: the first leaf (L1GT), second leaf (L2GT), and third leaf (L3GT). A multi-disciplinary approach was used to evaluate these samples. Computer vision and electronic tongue technology were used to quantitatively characterize the infusion color and taste intensity. Chemical composition was identified using ultraviolet-visible spectrophotometry, high-performance liquid chromatography, and liquid chromatography-mass spectrometry metabolomics. In vitro simulation was conducted to measure the antioxidant capacities via 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, plus hypoglycemic potential after alpha-glucosidase and alpha-amylase inhibition. The key metabolites were identified to regulate the taste differences among grades using dose-over-threshold (DOT). The results showed that the leaf maturity dominated the sensory attributes, physicochemical indices, and health-promoting functions. In sensory characteristics, the infusion greenness rose significantly from 6.79 to 8.66, whereas the yellowness declined sharply from 54.80 to 24.40, as the leaf maturity increased from L1GT to L3GT. Concurrently, the electronic tongue response values shared the synchronic downward trends; The bitterness (8.95), astringency (5.87), umami (19.45), and richness (11.61) of L1GT all significantly exceeded those recorded for L3GT (8.00, 4.63, 17.69, and 9.88), indicating the tender leaves with the markedly more robust and complex flavor profile. In terms of chemical composition, there was a significant decrease in the contents of tea polyphenols, free amino acids, and total flavonoids, particularly with the increasing maturity. While the soluble sugars and total chlorophyll increased to differentially regulate metabolite accumulation driven by leaf development. In vitro biological activity, both antioxidant capacities and enzyme inhibitory activities, displayed significant dose-dependent relationships with the leaf tenderness. The L1GT demonstrated the superior functional performance, with the lowest median inhibition concentration (IC₅₀) values for DPPH and ABTS radical scavenging at 14.16 and 239.40 µg/mL, respectively. Similarly, the L1GT manifested the most potent inhibition against α-glucosidase (IC₅₀=32.72 µg/mL) and α-amylase (IC₅₀=32.24 mg/mL). In the context of metabolic mechanisms, 24 differential compounds were identified after metabolomics analysis. Furthermore, 11 core substances were pinpointed as the critical contributors to taste formation via DOT screening. Notably, there was a significant increase in the abundances of L-glutamic acid, epigallocatechin gallate, epicatechin gallate, kaempferol-3-O-glucoside, kaempferol-3-O-rutinoside, myricetin-3-O-galactoside, gallic acid, and caffeine in the L1GT. Pathway analysis showed that the phenylpropanoid biosynthetic pathway, the tricarboxylic acid cycle, and the xanthine methylation were regulated for the intense bitterness, astringency, and umami characteristics of younger leaves. In conclusion, the maturity of fresh leaves served as the influencing factor to determine the sensory quality and health-promoting functions of Lu’an Guapian. Young leaves were characterized by the high concentrations of polyphenols, flavonoids, and amino acids, thereby combining potent antioxidant activity with a rich taste profile. Conversely, the mature leaves accumulated more sugars, thereby lacking the high biological activity in the tender shoots. The high-value utilization can also be realized for the fresh leaves at different maturity levels. This finding can provide a solid theoretical basis for the grading of Lu’an Guapian raw materials during processing.